Evaluation of the impact of successive acaricide treatments on resistance evolution in Rhipicephalus microplus populations: Monodrugs versus drug combinations.

The aim of this work was to comparatively evaluate the evolution of resistance in Rhipicephalus microplus tick populations exposed to successive treatments with monodrug-based formulations and combinations of them in the same commercial formulation. Thirty-six heifers, naturally infested with R. microplus, were divided into three groups (G) and subjected to three successive treatments, on days 0 (Nov-2021), 43 (Jan-2022) and 78 (Feb-2022), with the following formulations: I) ivermectin 3.15% (Ivomec Gold®) (GI), II) fipronil 1% (Ectoline®) (GII) and III) a combination of fipronil 2% and ivermectin 1% (Vaquero®) (GIII). From Nov-2021 to Dec-2022, counts of R. microplus were periodically performed to evaluate the tick infestation. Engorged females were collected at the beginning and end of the trial to determine the evolution of tick resistance to ivermectin and fipronil by in vitro bioassays. At the end of trial, GII and GIII had higher tick counts (39.18 ± 11.88 and 38.33 ± 14.31, respectively) than group I (5.11 ± 6.24) (P<0.05). The in vitro results shows that the resistance ratio (RR50) values after the treatments were higher for fipronil (5.584 and 5.649 for GII and GIII, respectively) than for ivermectin (1.165 and 1.088 for GI and GIII, respectively). In the group treated with the combination (GIII), the RR50 increased for both drugs simultaneously. These results suggest that the successive use of drug combinations could exacerbate the problem of multi-resistance of R. microplus to chemical acaricides.

Alternative applications of the strategic control against the cattle tick Rhipicephalus (Boophilus) microplus in a subtropical area.

Although different evaluations on the efficacy of the strategic control against Rhipicephalus microplus have been performed, the effects of successive applications of these schemes on the abundance of cattle ticks have not been evaluated. The aim of this work was to analyse the long-term effect of strategic applications of chemical acaricides on the R. microplus infestation in cattle in a subtropical area. These schemes are based on the application of three annual treatments between late winter and late spring. Additionally, a trial to evaluate the efficacy of the strategic control by deferring the first treatment from late winter to spring and the third treatment from late spring to summer was also carried out. The efficacy of the strategic control applied on 3 consecutive years was significant. The tick infestation in the treated group always remained at low levels, because mean number of ticks was almost never higher than 20. Regarding the trial where the third application of acaricide was deferred from spring to summer, and the first one from late winter to spring, the differences between treated and control group were significant in all post-treatment counts. The results of this study add evidence that support the sustainability of the strategic control in subtropical areas where the population dynamics of R. microplus is characterized by a well-marked seasonal pattern. Three relevant aspects were determined: (i) the feasibility and efficacy of successive applications of the strategic control in consecutive years; (ii) the time window to start the sequence of treatments is from late winter to mid-spring; (iii) it is achievable deferring the last treatment from late spring to summer if the tick infestation levels on cattle are low enough to allow it.

In vitro diagnosis of resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fipronil in Argentina.

The aim of this work was to adapt and apply the in vitro larval bioassay packet test (LPT) to diagnose resistance of Rhipicephalus microplus ticks to fipronil in Argentina. Ten serial dilutions of fipronil were included in the LPT bioassay. Seven populations of R. microplus from six Argentinean Provinces were evaluated. Samples from Colonia Benítez, Quimilí and Intiyaco were unambiguously classified in the category 'resistant'. In all these cases the 95% confidence intervals did not overlap with that of the susceptible reference strain and the slope values were lower than the slope value obtained for the susceptible reference strain. The resistance ratio (RR50) values of the populations from Colonia Benítez I and II, Quimilí and Intiyaco were 2.23, 3.49, 6.84 and 3.39, respectively. Populations from El Colorado I and II, Acambuco, Vera and Feliciano were classified as 'susceptible' because the 95% confidence intervals overlapped with that of the susceptible reference strain and the RR50 values never exceeded 1.32. The results of these LPT bioassays indicate the presence of fipronil resistant R. microplus populations in Argentina.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to ivermectin in Argentina.

Resistance to ivermectin in populations of the cattle tick Rhipicephalus microplus in Argentina was diagnosed in this work. The in vitro larval immersion test (LIT) was used to determine quantitatively the levels of resistance to ivermectin in different populations of R. microplus. Additionally, field trials to control natural infestations of R. microplus on cattle with a commercial formulation of ivermectin 3.15% were carried and jointly analyzed with the in vitro assays. The phenotypic response of the populations analyzed was not uniform. Five of them were classified as susceptible, four populations as resistant, and one in the category "incipient resistance". Regarding the field trials, the therapeutic efficacy in a population classified with LIT as susceptible achieved values higher than 94% two weeks after treatment, and no reproductively viable females were observed after the second day post-treatment. Conversely, the values of efficacy percentage in a population (named as "San Martín") classified with LIT in the category "incipient resistance" never exceeded the 70.8%, and engorged females were collected in practically all counts. The population "San Martín" was classified in the category "incipient resistant" with LIT analysis, but the field trial unambiguously shows that this tick population is resistant. The comparison of the results obtained with LIT in vitro assays and through field trials shows that biased estimations of resistance levels may occur when resistance ratios (RR) values are ≤2, and additional field efficacy trials could be needed to know with precision the status of the tick populations evaluated.